The interaction mechanisms of adhesion molecules between vascular endothelial cells and lymphocytes are well recognized in the view of immune responses. Only few reports, however, exist regarding pivotal roles of adhesion molecules in the movement of lymphocytes in the lymphatic system. In this study, we investigated interaction mechanisms of adhesion molecules between rat lymphatic endothelial cells and lymphocytes collected from rat lymph nodes. We have already established the cell line of lymphatic endothelial cells isolated from rat thoracic duct (Microcirculation, 10: 127-131, 2003). We used the cultured lymphatic endothelial cells and rat lymph nodes-derived lymphocytes labeled with 5-(and 6-) carboxyfluoresence in diacetate succinimidyl ester (CFSE) for visualization. After the co-incubation with the lymphatic endothelial cells and lymphocytes for 30 min at 37°C, nonadherent-lymphocytes were removed by washing with PBS three times. The binding ability of the labeled-lymphocytes was assessed by microscopic examination. A selective adhesion between the no-treated lymphatic endothelial cells and the lymphocytes was clearly observed in the control. In addition, pre-treatment the lymphatic endothelial cells with anti E-selectin antibody (30μg/ml) for 1 hr at 37°C caused a significant decrease of numbers of the lymphocytes adhered selectively with the cultured lymphatic endothelial cells. This finding suggests that E-selectin may play an important role in the adhesive mechanisms between lymphatic endothelial cells and lymphocytes. [Jpn J Physiol 54 Suppl:S93 (2004)]

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Gravity acts on the circulatory system to decrease arterial blood pressure (AP) by causing blood redistribution and reduced venous return. To evaluate roles of the baroreflex and vestibulosympathetic reflex (VSR) in maintaining AP during gravitational stress, we measured AP, heart rate (HR), and renal sympathetic nerve activity (RSNA) in four groups of conscious rats, which were either intact or had vestibular lesions (VL), sinoaortic denervation (SAD), or VL plus SAD (VL+SAD). The rats were exposed to 3G in dorsoventral axis by centrifugation for 3min. In rats in which neither reflex was functional (VL+SAD group), RSNA did not change, but the AP showed a significant decrease (-8±1 mmHg vs. baseline). In rats with a functional baroreflex, but no VSR (VL group), the AP did not change and there was a slight increase in RSNA (25±10% vs. baseline). In rats with a functional VSR, but no baroreflex (SAD group), marked increases in both AP and RSNA were observed (AP 31±6 mmHg and RSNA 87±10% vs. baseline), showing that the VSR causes an increase in AP in response to gravitational stress; these marked increases were significantly attenuated by the baroreflex in the intact group (AP 9±2 mmHg and RSNA 38±7% vs. baseline). In conclusion, AP is controlled by the combination of the baroreflex and VSR. The VSR elicits a huge pressor response during gravitational stress, preventing hypotension due to blood redistribution. In intact rats, this AP increase is compensated by the baroreflex, resulting in only a slight increase in AP. [Jpn J Physiol 54 Suppl:S93 (2004)]

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Chronic global hypoxia causes sustained pulmonary hypertension of which an impairment of the pulmonary endothelial nitric oxide (NO) pathway has been implicated as a contributing factor. Within the CNS, NO also acts as a sympathoinhibitory substance, so that NO inhibition (e.g. using the NOS antagonist L-NAME) increases sympathetic outflow and ultimately arterial blood pressure. The effect of CNS NO inhibition on pulmonary vasculature is unknown. Thus, we hypothesised that the CNS NO may help modulate pulmonary vascular tone and act to attenuate the magnitude of pulmonary hypertension during chronic hypoxia. Male Sprague Dawley rats received a continuous intracerebroventricular infusion of either L-NAME (150 μg/kg/day) or vehicle (aCSF). Pulmonary arterial pressure (PAP) was measured in conscious rats using a pressure telemetric transmitter. One week after surgery, rats were exposed to chronic hypocapnic hypoxia (12% O₂) for 2 weeks. As expected, chronic hypoxia significantly increased PAP. However, the central administration of L-NAME did not appear to alter the magnitude of the chronic hypoxia-induced pulmonary hypertension. These results suggest that the central inhibition of NO may not exacerbate the increase in sympathetic outflow and, thus, may not influence the pathomechanisms responsible for pulmonary hypertension during chronic hypoxia. In further experiments we will determine whether increasing central NO production (L-arginine infusion) attenuates the development of pulmonary hypertension during chronic hypoxia. [Jpn J Physiol 54 Suppl:S93 (2004)]

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Heart rate (HR) is affected by physical activity (PA) and mental conditions during awake and sleep, both of which produce a circadian rhythm of the HR. Numerous studies reported relationships between HR and PA. However, little is known about the method to evaluate mental stress based on the HR-PA data in free-moving humans. The aims of the present study were to examine relationships between HR and PA and to find a clue to assess mental stress out of the relations. Subjects were a healthy adult woman and 5 men. Most of them were hospital workers. Continuous recordings of HR and PA were performed for 24 hours or more by using a portable monitor (Active Tracer AC-301, GMS Inc., Japan). PA was assessed by the number of counts beyond an acceleration of 0.02G. Physical behaviors and mental status were briefly described on sheets or recorded by an IC-voice recorder. In most cases during sleeping and awake, HR had a significant correlation with PA due to turning over in bed. In the subjects who did not feel any remarkable stress during daytime including working hours, logarithm plots of HR vs. PA exhibited a statistically significant linear correlation. However, in a subject who experienced mental stress during his work, frequent increases in HR with low levels of PA produced a prominent variability in the logarithm plot of HR-PA. HR-PA plot may be avaiable for the evaluation of such non-physical stress as mental one. [Jpn J Physiol 54 Suppl:S94 (2004)]

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We analyzed components of pressure waves in the atherosclerotic aorta and determined augmentation index (AIx) of the waves in Kurosawa and Kusanagi-Hypercholesterolemic (KHC) rabbits. Pressure and flow waves were simultaneously recorded at the ascending aorta under pentobarbital anesthesia in 17 KHC and 17 normal rabbits aged 10-12 months old. The time at which the 2nd zero crossing from above to below in the 4th derivative of the pressure wave in each cardiac cycle was estimated as a peak of early systolic waves. Pulse pressure was taken as an amplitude of late systolic waves. AIx was determined as an amplitude ratio of the late to the early pressure waves. Systolic (SAP), diastolic (DAP) and mean (MAP) pressures, amplitude of the late systolic waves, AIx and total peripheral resistance (TPR) were significantly greater in KHC rabbits than in control rabbits, respectively, whereas the early systolic waves were significantly smaller in KHC rabbits than in control rabbits. There were no significant differences in ascending aortic flow and stroke volume (SV) between the two rabbit groups. AIx showed significant correlations with SAP, DAP, MAP, TPR and SV in KHC rabbits, respectively, while it did not correlate with total percent lesioned area. In control rabbits, AIx showed no correlation with them. The significantly increased AIx in KHC rabbits was considered to be due mainly to the decreased early and increased late systolic waves. [Jpn J Physiol 54 Suppl:S94 (2004)]

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[Introduction] It is known that chronic infusion of isoproterenol induces cardiac hypertrophy. The aim of the present study was to characterize left ventricular (LV) function in hypertrophied rat hearts. [Methods] Delivery of drug was achieved by implanting osmotic minipump subcutaneously in the neck of rat. Either isoproterenol (1.2 or 2.4 mg/kg/day for 3 days: Iso 1.2 and Iso 2.4), metoprolol (24 mg/kg/day for 3 days) + Iso 1.2 (Meto+Iso) or vehicle (saline 2.4 ml/kg for 3 days: Sa) was infused subcutaneously. Osmotic minipump was removed three days after the implantation. One hour after removal of the minipump in Sa, Iso 1.2, 2.4 and Meto+Iso groups and two days after removal of the minipump in Iso 1.2 group [ Iso (-) ], we recorded continuous LV pressure-volume (P-V) loops of in situ ejecting hypertrophied rat hearts. Curved LV end-systolic P-V relation (ESPVR) and systolic P-V area at midrange left ventricular volume (PVA_mLVV) was obtained from a series of LV P-V loops in Sa and Iso groups. PVA_mLVV has been proposed a good mechanoenergetic index to evaluate LV function. [Result] PVA_mLVV in Iso 1.2 and Iso 2.4 groups were significantly smaller than that in Sa group. PVA_mLVV in Iso (-) group showed the same level as that in Sa group. PVA_mLVV in Meto + Iso group was significantly larger than that in Iso 1.2 group. The changes in PVA_mLVV in isoproterenol-induced reversible hypertrophied rat hearts, mediated by β1-receptor, precisely reflect the changes in LV work capability even in remodeling rat hearts. [Jpn J Physiol 54 Suppl:S94 (2004)]

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The ductus arteriosus (DA), a fetal connection between the pulmonary artery and the aorta, plays an important role in in utero cardiovascular circulation. Prostaglandin E (PGE) plays a principal role in maintaining the patency of the DA. The DA displays the higher sensitivity to PGE than other vascular smooth muscles. PGE activates adenylyl cyclases (ACs) via the EP4 receptor, resulting in an increase in the intracellular cAMP in the DA. ACs consist of 9 isoforms that demonstrate distinct tissue distribution and function. However, characterization of ACs remain undetermined in the DA. We examined the expression of AC isoform mRNAs in the DA by semi-quantitative and quantitative RT-PCR. Pooled tissues of the DA were obtained from Wistar rat embryos at embryonic day19 and day21, and neonates on the day of birth. AC2, AC3, AC4, AC5, AC6, AC7, and AC9 were expressed in the DA, while AC1 and AC8 were not detected. The expression of AC2, AC4, and AC6 mRNAs in the DA were significantly higher than that in the aorta. In particular, AC2 was expressed to a strikingly higher degree in the DA at embryonic day21 and the day of birth (~ 30% increase) than in the adult aorta. The expression of AC3, AC5, and AC7 mRNAs in the DA were comparable with those in the aorta. The present study identified multiple AC isoforms in the rat DA. AC2, AC4 and AC6 isoforms, in particular AC2, may play an important role in mediating PGE signal in the DA. [Jpn J Physiol 54 Suppl:S94 (2004)]

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We recently demonstrated that the baroreflex bradycardia evoked by stimulation of the aortic depressor nerve (ADN) is attenuated immediately before or at the onset of voluntary static exercise in conscious cats, whereas the ADN stimulation-induced depressor response remains the same (Komine et al. Am J Physiol, H516-526, 2003). This result suggested that central command blunts the cardiac component, but not vasomotor component, of the aortic baroreflex at the onset of voluntary static exercise. The aim of the present study was to examine whether nitric oxide (NO) contributes to the inhibition of the cardiac component of the aortic baroreflex by central command, by administering a synthesis inhibitor of NO (L-NAME, 5mg/kg i.v.) using conscious cats. When the ADN was stimulated at rest, heart rate decreased by 36±6 beats/min and arterial blood pressure reduced by 10±2 mmHg. The ADN stimulation-induced bradycardia was attenuated at the onset of exercise to 63±8% of the preexercise bradycardia. Following L-NAME, the reduction of the baroreflex bradycardia seen at the onset of exercise was absent. This finding suggested that L-NAME blunted the attenuating effect of central command on the ADN stimulation-induced bradycardia. Therefore it is likely that endogenous nitric oxide released in the brain plays a role in the inhibition of the cardiac component of the aortic baroreflex by central command at the onset of voluntary static exercise. [Jpn J Physiol 54 Suppl:S95 (2004)]

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We determined whether exercise pressor reflex (sensory afferent input from muscle to the medulla) decreases vascular conductance in an inactive contralateral limb as to increase blood flow to the active muscle. Sprague Dawley rats (n=9) were instrumented with electro-magnetic flow probe (MFV2100, 1mm probe; Nihon-Koden) on left iliac artery and a catheter in carotid artery. Rats were decerebrated at midcollicular level. Right triceps surae muscles were exposed and the Achilles tendon was ligated. The tendon was stretched at 300 g for 30 s to stimulate the muscle. The vascular conductance (VC) of left inactive leg was calculated from mean arterial pressure (MAP) and iliac blood flow. The stretch significantly increased the MAP (+20±3％, peak increase from baseline; mean±SEM), while it significantly decreased the VC in the left iliac artery (–9±1％, average over stimulation period). After cutting the left sciatic nerve, the stretch significantly increased MAP (+13±3％, peak), but did not significantly change the VC in the inactive left iliac artery (+5±1％, average). This result suggests that exercise pressor reflex induces sympathetic vasoconstriction in the inactive contralateral muscle. [Jpn J Physiol 54 Suppl:S95 (2004)]

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Model-guided optimal treatment of hemodynamics is indispensable to improve outcome of acute heart failure patients. We constructed a comprehensive cardiovascular model by extending Guyton's model. The model enabled one to describe pathophysiology of unilateral left heart failure, often accompanying myocardial infarction. Venous return (VR) property was described by a surface instead of a curve, as a function of atrial pressures (PLA, PRA) as VR =V/W-GRPRA-GLPLA, given blood volume (V). The pumping ability of hearts was expressed by a cardiac output (CO) curve in 3D space, as CO=SL[ln(PLA-BL)+CL] =SR[ln(PRA-BR)+CR], given slopes of CO curve (S's). W, G's, B's and C's are parameters. Methods: Parameters W, G's, B's and C's were found to be relatively constant. In 8 dogs with/without acute heart failure, we determined V and S's from a single set of CO, PRA, PLA and standard parameter values. We then altered V from -8 to +8ml/kg and examined if predicted hemodynamics agreed those measured. Results: Standard parameters were identified as W(0.13), GR(19.6), GL (3.5). BL(2.1), CL(1.9), BR(2.0) and CR(0.80). Using these, we could accurately predict CO (y=0.93x+6.5, r2=0.96), PRA (y=0.87x+0.4, r2=0.91) and PLA (y=0.90x+0.48, r2=0.93). Conclusion: The proposed framework enabled one to accurately predict hemodynamics after volume intervention from a clinically available measurement, a single set of CO, PRA and PLA, and to optimize treatment of heart failure. [Jpn J Physiol 54 Suppl:S95 (2004)]

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It has been assumed that ventricular excitation rhythm follows atrial excitation rhythm during dynamic exercise in humans. To examine whether this assumption is true, we measured electrocardiogram (ECG) and analyzed the beat-to-beat changes in PP, PR and RR intervals before, during, and after dynamic exercise in 11 sedentary and 9 trained subjects. Stair-stepping exercise was performed for 10 min in the upright posture. In sedentary subjects, the mean value of RR intervals before exercise was the same as that of PP interval (650±65ms) and the mean PR interval was 163±19ms. RR interval decreased during exercise to 378±21ms similar to PP interval, and PR interval also decreased to 145±11ms. Although the standard deviation (SD) of RR intervals before exercise coincided with SD of PP intervals, it became smaller during exercise than SD of PP intervals. On the contrary, SD of PR intervals was increased during exercise from 8.6±2.3ms to 9.0±3.4ms. The similar results were observed in the trained subjects. These findings indicated that beat-to-beat ventricular excitation rhythm does not simply follow atrial excitation rhythm during dynamic exercise, although the average value of RR intervals over a certain period is always the same as that of PP intervals. We conclude that ventricular excitation rhythm seems to be more stable during exercise than atrial excitation rhythm, because PR interval changed during exercise so as to cancel an alteration in the preceding PP interval in humans. [Jpn J Physiol 54 Suppl:S95 (2004)]

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Objective: The purpose of present study was to determine whether ischemic preconditioning (PR) or ischemic postconditioning (PT) could protect isolated perfused hearts from hydrogen peroxide(H₂O₂)-induced damage. Methods:Male SD rats were used. Langendroff-perfused rat hearts were subjected to 1) Control(CT) group: 30 min 100 μM H₂O₂ perfusion followed by 30 min washout. 2) PR group: 3 times ischemic preconditioning (3 min ischemia and 5 min reperfusion), followed by 30 min 100 μM H₂O₂ perfusion and 30 min washout. 3)PT group: 30 min 100 μM H₂O₂ perfusion followed by 3 times ischemic postconditioning (30 sec ischemia and 30 sec reperfusion) and 30 min washout. Results:After 30 min perfusion with H₂O₂, left ventricular developed pressure (LVDP) of CT, PR and PT groups decreased to 29.5±2.8%, 40.8±5.1% and 35.0±5.1%, respectively. The decrease of LVDP in PR group was significantly (P<0.05) lower than those of CT and PT groups. AT the end of washout period , LVDP of PR and PT groups recovered to 95.2±6.6% and 96.3±2.3% respectively which were significantly better than that of CT group (75.7±7.5%,P<0.05). Recovery of LVDP was quickest in PT group indicating protective mechanism different from that of PR. Conclusions:The results of this study indicate that ischemic preconditioning and postconditioning rapidly promote LVDP recovery and attenuate H₂O₂-induced ventricular dysfunction. [Jpn J Physiol 54 Suppl:S96 (2004)]

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Purpose: Protective effect of preconditioning (PR) and postconditioning (PT) for cardiac hypoxia-reoxygenation was examined. Methods: Hearts from male SD rat were Langendorff perfused and were made hypoxic for 30 min and reoxygenated for 30 min. PR was 3 times 5 min ischemia-3 min reperfusion cycle before hypoxia. PT was 3 times 30 sec ischemia-30 sec reperfusion cycle before reoxygenation. PR+PT group recieved both protocols. LV developed pressure (LVDP) and heart rate (HR) were monitored and as an index of irreversible damages, GOT release was quantified. Results: The recovery of pressure rate product (PRP; LVDP×HR) by reoxygenation was 19.7±4.6% in control group (CT) at 5 min. The recovery was significantly facilitated in PR and PT; PRP reached 33.0±10.2 and 41.0±7.5%, respectively at 5 min. PR+PT showed further quick recovery reaching 46.8±5.9% at 5 min. The recovery of PRP at 30 min reoxygenation in CT was 57.5±2.3%, while it recovered to 67.1±5.3% in PR and 63.9±4.4% in PT. However, in PR+PT, it remained 49.7±6.1%. GOT release (IU/min/g) at 2 min reoxygenation was 1.52±0.15 in CT. In PR (1.04±0.26) and PR+PT (1.15±0.14), increase in GOT was significantly (P<0.05) suppressed but there was no significant difference between PT (1.64±0.30) and CT. Conclusions: Either ischemic PR or PT showed cardiac protection against hypoxia-reoxygenation. However, in PR+PT group, protective effect disappeared. These results suggest that the mechanism of protection by PT is differ from that of PR, and their protective effects would be inhibited by each other. [Jpn J Physiol 54 Suppl:S96 (2004)]

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It is well known that extracellular acidosis modulates many types of ion channels in excitable and non-excitable cells. Recently, a novel Cl⁻ current that is activated by acidic pH has been found in rat sertoli cells. In the present study, we identified a similar current (I_Cl.pH) in ventricular myocytes from mouse and guinea-pig. When acidic solution was applied to the cells, I_Cl.pH appeared with a delay of ~1 min, increased gradually, and reached a maximum in ~5 min. In contrast, the current disappeared rapidly (<1 min) upon resumption of the solution with normal pH (7.4). I_Cl.pH was activated in a pH-dependent manner, with a half maximal activation at about pH 6.0. I_Cl.pH exhibited a weak time-dependent activation, the current amplitude slightly increasing during depolarizing step pulses. I-V relationship of I_Cl.pH showed a strong outward rectification under symmetrical [Cl⁻] conditions. The anion selectivity of this current was estimated to be I⁻ > Cl⁻ > Asp⁻. Pharmacological studies showed that I_Cl.pH was inhibited by several Cl⁻ channel blockers (DIDS, niflumic acid and glibenclamide). Thus, the properties of I_Cl.pH differ from those of other cardiac Cl⁻ currents (volume-regulated Cl⁻ current, inwardly rectifiying Cl⁻ current, Ca²⁺-activated Cl⁻ current or CFTR current). I_Cl.pH may play a role in the control of the action potential duration under pathological conditions, such as ischemia-related cardiac acidosis. [Jpn J Physiol 54 Suppl:S96 (2004)]

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In ventricular myocytes, α₁-adrenoceptor stimulation (α₁ARS) increases L-type Ca²⁺ current (I_Ca,L) in perforated patch clamp (PP) recording but not in conventional whole cell recording. We hypothesized Ca²⁺-related protein kinase such as Ca2+/calmodulin-dependent protein kinase II (CaMKII) or protein kinase C (PKC) is involved in the regulation of I_Ca,L during α₁ARS. In this study, we investingated the effect of phenylephrine (Phe) on I_Ca,L in adult rat ventricular myocytes using PP recording. As previous studies, we observed biphasic modulation in I_Ca,L by 10 μM Phe; a transient decrease followed by a sustained increase. However, 1 μM Phe just increased I_Ca,L. In the presence of CaMKII inhibitor 0.5 μM KN-93, a CaMKII inhibitor, 10 μM Phe produced only a sustained negative phase of I_Ca,L while 1 μM Phe did not alter I_Ca,L. In the presence of 10 μM chelerythrine, a PKC inhibitor, 10 μM Phe produced only a sustained negative phase as in the presence of CAMKII inhibitor. Our result suggests that (1) the effect of α₁ARS on I_Ca,L can be classified into two phases (negative phase and positive phase) which have different concentration-dependence and (2) CaMKII and PKC regulate the positive phase of I_Ca,L during α₁ARS. [Jpn J Physiol 54 Suppl:S96 (2004)]

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It is generally known that α₁-adrenoceptor stimulation shows a positive inotropic effect on mammalian myocardium. In mouse myocardium, however, we reported that an α₁-adrenoceptor stimulation showed a negative inotropic effect. In the present study, we explored the mechanism of the negative inotropic effect in mouse myocardium. We used isolated ventricular papillary muscles of mouse and simultaneously measured isometric tension and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) using the aequorin method. In twitch contraction, phenylephrine (Phe) dose- dependently (1~100 μM) decreased the Ca²⁺ transient and tension. To estimate the Ca²⁺ sensitivity, tetanic contraction was produced and the relation between [Ca²⁺]_i and tension at the steady-state was measured. Phe (10 μM) decreased the Ca²⁺ sensitivity and this effect was completely blocked by prazosin (3 μM). Phorbol 12- myristate 13-acetate (PKC activator, 1 μM) decreased the Ca²⁺ transient and the Ca²⁺ sensitivity, which was almost the same as that of Phe. After PKC activation, the negative inotropic effect of Phe was not observed. The negative inotropic effect of Phe was completely blocked by calphostin C (PKC inhibitor, 1 μM). These results suggest that the negative inotropic effect of Phe in mouse myocardium is due to the decrease in the Ca²⁺ transient and the Ca²⁺ sensitivity. The activation of PKC pathway is involved in the negative inotropic effect of α₁-adrenoceptor stimulation in mouse myocardium. [Jpn J Physiol 54 Suppl:S97 (2004)]

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It is well known that the low-frequency oscillations (<0.6 Hz) in blood pressure reflect sympathetic activity to the resistance vessels and that the nitric oxide (NO) system is a potent mechanism in buffering these blood pressure oscillations of central origin. In the present study, the buffering effect of NO on sympathetic activity was investigated by power spectral analysis of arterial pressure in conscious normotensive rats. Ganglion blocker hexamethonium bromide (C6), N^ω-nitro-L-arginine methyl ester (L-NAME), and L-arginine (L-Arg) were used to examine the hemodynamic effects of the NO system and the sympathetic nervous system on mean arterial pressure (MAP), mean heart rate (MHR), arterial pressure variability (APV) and heart rate variability (HRV). APV (<0.25 Hz) increased with the significant increase of MAP after administration of L-NAME, while APV (0.25~0.6 Hz) decreased. In addition, elevated APV (<0.25 Hz) decreased with the significant decrease of MAP by addition of L-Arg, while APV (0.25~0.6 Hz) increased. Oscillations <0.6 Hz of APV were eliminated by C6 and C6 + prazosin (α₁-blocker) and MAP decreased by 20 mmHg. MHR decreased by L-NAME and increased by L-Arg. HRV (<0.6 Hz) reflected sympathetic activity to the heart. These results suggest that, (1) APV (<0.6 Hz) is originated from the sympathetic nervous system, (2) the buffering effect of NO on APV is below 0.6 Hz and the effect of NO on APV (0.25~0.6 Hz) is exerted via baroreflex, and (3) NO interacts with sympathetic activity by reducing the Ca²⁺ concentration in smooth muscle cells. [Jpn J Physiol 54 Suppl:S97 (2004)]

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It is well known that intracellular Ca²⁺ regulates cardiac muscle contraction and cardiac Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA2a) plays a dominant role in Ca²⁺ handling. During acidosis, peak tension decreases, although the peak of the Ca²⁺ transient (CaT) increases, which could be due to a decrease in the Ca²⁺ sensitivity of the myofilaments. We hypothesized that an increase in the SERCA2a activity delivers more Ca²⁺ to the contractile elements and diminishes the decrease in tension during acidosis. In order to prove the hypothesis, we used the aequorin method and measured tension with CaT in left ventricular papillary muscles obtained from the transgenic mouse (TG) which overexpresses cardiac-restricted SERCA2a (almost doubled) and from the non-transgenic littermates (NTG). The peaks of CaT and tension in TG were larger than those in NTG in the control solution. In CO₂ (15%) acidosis, tension decreased to 25% of the control in NTG and decreased to 50% in TG. During recovery from acidosis, tension did not reach the control level in NTG but fully recovered in TG. During acidosis and recovery phase, the peak of CaT in TG was significantly larger than that in NTG. These results suggest that the increased activity of SERCA2a could be beneficial to preserve tension during acidosis and recovery by delivering suffcient Ca²⁺ to the myofilaments. [Jpn J Physiol 54 Suppl:S97 (2004)]

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Cardiac muscle contraction is controlled by intracellular Ca transients (CaT) and muscle length. In the present study, we observed the effects of Ca delivery to contractile elements and muscle length on the recovery of contraction after CO₂ acidosis. We used the left ventricular papillary muscle obtained from C57BL/6 mice and from SERCA2a-overexpressed mice. We simultaneously measured CaT and tension in both preparations during and after CO₂ acidosis using the aequorin method (30°C).The length of the preparation was changed from Lmax, in which developed tension reached maximum, to 92%Lmax and 84%Lmax. At each length, tension decreased and CaT increased in CO₂ acidosis. After CO₂ acidosis, tension and CaT did not fully recover at Lmax. However, at shorter lengths, tension and CaT after CO₂ acidosis were well maintained compared to those before acidosis. We also measured CaT and tension at Lmax after CO₂ acidosis using SERCA2a-overexpressed transgenic myocardium (TG). In TG, CaT was significantly higher than that in non-TG during and after CO₂ acidosis. After acidosis, tension and CaT were well recovered compared to those in non-TG myocardium. Thus, sufficient Ca delivery to the myofilaments and muscle length are important factors for the recovery from pathophysiological conditions, in particular from CO₂ acidosis. [Jpn J Physiol 54 Suppl:S97 (2004)]

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We have optically mapped the spatiotemporal patterns of the spread of excitatory waves during "tachycardia-like excitation" in the isolated rat atrial preparation. The tachycardia-like excitation with a regular cycle length of 250 - 450 ms was evoked by regular pacing (0.5-1 Hz) applied with a bipolar electrode. The events lasted for various time periods (a few sec - longer than 10 min) and often terminated spontaneously. Multiple-site optical recording methods, using a voltage-sensitive merocyanine-rhodanine dye (NK2761) and a multi-element (12 × 12 or 16 × 16) photodiode array, were employed to simultaneously monitor action potentials from many sites in the atrium. The spread of excitation was assessed optically, and we mapped the conduction patterns of the excitatory waves. These maps indicate that, during the event of tachycardia-like excitation, the excitatory waves often propagated in a quasi-circular pathway which surrounded "blocked areas" including the ostium of the superior vena cava, suggesting the establishment of the macro re-entry. The blocked area was generated by the stimulation pulse applied shortly after the spontaneous excitation, and then the excitatory wave started to rotate around the blocked area. The blocked area was often maintained during the event of tachycardia-like excitation. In some cases, instable blocked areas were also observed. [Jpn J Physiol 54 Suppl:S98 (2004)]

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Increase of membrane potential (hyperpolarization) ruptures membrane as has been demonstrated by irregular inward currents (I_hi) and nuclear ethidium fluorescence increase (EB_fluo) in cardiac myocytes. We found that La³⁺ depresses I_hi. Polyethyleneglycol (PEG) is known to plug some large ion channels. Here, we applied La³⁺ and PEG (MW, 4,000) together or independently to isolated rabbit ventricular myocytes to examine their closing effects on electrically produced pores. The time integral of whole-cell I_hi during 40 s pulses (Q_hi) and EB_fluo during these pulses were estimated at membrane potentials between –80 and –160 mV. Q_hi was depressed by La³⁺ (30, 100, 1,000 nM) to about 15% at each potential and this depression was not affected by PEG. In contrast to La³⁺, Q_hi was little affected by 2 and 5% PEG in control. However, Q_hi was suppressed by 2 and 5% PEG in the presence of 1 mM-La³⁺. EB_fluo was depressed by La³⁺ maximally at 30 nM among above concentrations to 61% in control and to 42%in the presence of 5%-PEG. PEG (5%) voltage-independently depressed EB_fluo to 50% in control and the depression was increased by 0.1 mM- La³⁺ to 26%. It was suggested that La³⁺ mainly closes pores with small diameter, while PEG selectively closes those with larger diameter. La³⁺ could seal the pores by binding to their outer end and PEG could cooperatively enhance closing action of La³⁺ in the presence of high concentrations of La⁺. In addition, the results indicate that hyperpolarizations produce small pores with additional small number of ethidium⁺-permeable large pores. [Jpn J Physiol 54 Suppl:S98 (2004)]

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It is thought that the initial increase in heart rate (HR) at the onset of dynamic exercise is caused by rapid withdrawal of cardiac parasympathetic tone, but not by an increase in sympathetic nerve activity. The M₂ receptor (of M₁-M₅ muscarinic receptors) is believed to be predominantly expressed in cardiac muscle. Therefore, we examined the role of M₂ receptor in HR control during voluntary wheel running exercise using M₂-KO mice. HR was determined by electrocardiogram telemetry in conscious mice. M₂-KO mice showed slight tachycardia and more diminished respiratory oscillations in HR than wild type (WT) mice at rest. Muscarinic agonist bethanechol induced marked vagal bradycardia and respiratory oscillations in HR in WT, but not in M₂-KO mice. With the onset of exercise, HR increased rapidly and remained elevated throughout exercise. This initial HR increase was blunted by a β-adrenergic blocker propranolol in both groups. A gradual increase in respiratory oscillation of HR observed in WT was absent in M₂-KO mice after exercise cessation. These results indicate that vagal bradycardia and the respiratory sinus arrhythmia in the inactive state are chiefly mediated by the M₂ receptor, whereas cardiac acceleration at the onset and during dynamic exercise is not solely mediated by it. In conclusion, the parasympathetic component plays a more important role in lowering HR from activated levels. [Jpn J Physiol 54 Suppl:S98 (2004)]

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Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated a time-dependent expression of ion channels and signal transduction pathways. However, Na⁺/Ca²⁺ exchangers (NCX), which play crucial roles for cardiac functions, have not been well elucidated. In this study, we investigated the functional expression of NCX in mES cells at various stages of in vitro differentiation into cardiomyocytes. When NCXs were blocked by 100 μM KB-R7943, the dynamic changes in [Ca²⁺]_i were observed in almost all mES cells, which showed two different patterns, namely a [Ca²⁺]_i transient and a sustained elevation of [Ca²⁺]_i. A [Ca²⁺]_i transient was observed in 90% of undifferentiated mES cells but few derived cardiomyocytes. In contrast, a sustained elevation of [Ca²⁺]_i was observed in derived cardiomyocytes and [Ca²⁺]_i increased in a time course of differentiation. By RT-PCR the expression levels of a NCX1 mRNA increased during the differentiation. When Na⁺ pumps were blocked by ouabain, [Ca²⁺]_i oscillations could be induced in 75% of derived cardiomyocytes at the middle stage of differenitation but not at the earlier stages, indicating the functional coupling of NCXs with Na⁺ pumps. Taken together, we conclude that the contribution of NCXs to lowing [Ca²⁺]_i become greater during differentiation and the functional coupling with Na⁺ pumps is established at the middle stage of the differentiation. [Jpn J Physiol 54 Suppl:S98 (2004)]

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It is known that the amplitude of the outward current of the strong inward rectifier K⁺ current, I_K1, in the atrium is smaller than that in the ventricle. However, the mechanism underlying the difference is not understood. Using the amphotericin B perforated-patch recordings, we found that the inward rectification of I_K1 in the atrial cells of the guinea-pig heart was stronger than that in the ventricular cells. Further, the outward transients of I_K1, which were elicited on repolarization in the ventricular cells, were hardly observed in the atrial cells. When the genes encoding the strong inward rectifier K⁺ channels Kir2.x, known to be expressed in the cardiac cells, were expressed in L cells, the inward currents of the Kir2.1 and Kir2.2 channels were similar to those of I_K1, while those of the Kir2.3 channels were different with respect to their significantly slower activation and strong sensitivity to the external pH. When the outward currents of Kir2.1 and Kir2.2, known to be regulated by the blockage of the channel by cytoplasmic polyamines and Mg²⁺, were recorded from the inside-out patches in the co-presence of 1 mM Mg²⁺ and 5-10 μM spermine, marked transient currents were observed on repolarization. The amplitude of the transients became minimal when the concentrations of spermine and spermidine coexisting with Mg²⁺ were increased to a level higher than ~30 μM. Our results suggest that the difference in I_K1 between the atrium and the ventricle is at least partly caused by higher concentrations of free polyamines in the atrial cells than in the ventricular cells. [Jpn J Physiol 54 Suppl:S99 (2004)]

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Central sympathetic activation is one of the mechanisms of salt-sensitive hypertension and reactive oxygen species (ROS) has been proposed to play a key role in it. We examined whether adrenomedullin (AM), an antioxidant peptide, might contribute to the central regulation of arterial pressure (AP). Four groups of mice were used: AM-knockout mice (AM(+/-)) and wild-type littermates (AM(+/+)), which were fed with normal or high salt diet. In urethane-anesthetized mice, intracerebroventricular infusion of NaCl increased AP and sympathetic nerve activity (SNA) in a dose-dependent fashion. The elevation of AP and SNA in salt-loaded AM(+/-) was significantly greater than those in the other three groups (p<0.05). In freely moving mice, similar exaggerated hypertension was observed in salt-loaded AM(+/-). Pretreatment with a membrane-permeable superoxide dismutase mimetic, Tempol, completely blocked the responses in both AP and SNA, indicating participation of ROS. NaCl-induced ROS production in the hypothalamus was significantly greater in salt-loaded AM(+/-) than in AM(+/+), as determined by chemiluminescence assay. AM contents in the brain from AM(+/+) mice were significantly elevated by salt loading, whereas they were not elevated in AM(+/-) mice. These results suggest that AM in the brain inhibits sympathetic activation in salt-induced hypertension through its antioxidant effect. [Jpn J Physiol 54 Suppl:S99 (2004)]

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It is well known that superoxide production from aorta increases with progress of hypertension. However, amounts of superoxide production may differ from organ to organ with progress of hypertension. In this study, superoxide released from isolated organs of short-term hypertensive rats was measured by lucigenin-derived chemiluminescence (LDCL). Spontaneously hypertensive rats were used as a hypertensive model. These rats were fed with high salt (8% NaCl) diet for 6 weeks from the age of 8 weeks. Aorta, kidney, heart and liver were isolated from each rat. LDCL of superoxide released from these tissues was measured using a luminometer. Systolic blood pressure remarkably increased after 6 weeks (161 ± 13 (0 weeks); 275 ± 9 (6 weeks) mmHg, p < 0.05). In aorta, superoxide production increased significantly (316 ± 95 (0 weeks); 630 ± 107 (6 weeks) counts/min/mg of dry tissue, p < 0.05). Superoxide production from kidney also increased (881 ± 249 (0 weeks); 1192 ± 250 (6 weeks) counts/min/mg). However, in heart and liver, superoxide production did not change (heart: 466 ± 132 (0 weeks), 441 ± 133 (6 weeks); liver: 1689 ± 328 (0 weeks), 1672 ± 395 (6 weeks) counts/min/mg). In conclusion, it is suggested production and scavenging systems of superoxide in aorta and kidney are influenced even after short-term hypertension while those in heart and liver are not. [Jpn J Physiol 54 Suppl:S99 (2004)]

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We investigated the endothelial function and oxidative stress in Dahl salt-sensitive rats with risk factors for atherosclerosis, i.e. hypertension and/or hypercholesterolemia. 109 rats were randomly assigned to four experimental diet groups; control (MF), high-salt (S), high-cholesterol (C), and combination of high-salt and high-cholesterol (SC), taking these diets ad libitum for 8 weeks. After physiological examination every other week, we observed the distribution of NO production and eNOS expression in endothelial cells around aorto-renal bifurcation of rats by using confocal laser scanning microscopy (LEICA TCS NT), and also measured plasma NOx and superoxide production in each organ. NO production was suppressed at the cranial portion of aorto-renal bifurcation regardless of diet groups. Plasma NOx was increased in the risk factor groups. Superoxide production increased in kidney and abdominal aorta in SC. In groups C and SC fatty liver was observed, and in groups S and SC cardiac hypertrophy and renal hypertrophy was induced. In risk factor groups, NO production was suppressed at the cranial portion of aorto-renal bifurcation, which is atheroma prone portion, same as a control group. Therefore local suppression of NO production in risk factor groups contributed to the localization of atherosclerosis. We conclude that NO and superoxide might be involved in the initiation and development of atherosclerosis. [Jpn J Physiol 54 Suppl:S99 (2004)]

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Action potential duration (APD) of cardiac myocytes are determined by the balance of inward (depolarizing) and outward (hyperpolarizing) currents. In principle, outward currents contribute to shorten and inward currents contribute to prolong APDs. There are cases contradictory to this rule, however, when currents were modified at the early stage of action potentials. We therefore analyzed the effects of inward/outward currents given at this critical phase, using several ventricular action potential models. In classical Beeler-Reuter model (1977), injection of inward current during initial 50msec of action potential shortened, while outward current prolonged APDs. This was primarily produced by the difference of voltage-dependent activation of delayed outward current (undivided I_K). The "reversed" effects of current injection on APD were observed also in Luo-Rudy model (1994). Here, not only different levels of I_Ks activation, but also the amount of Ca²⁺ influx and the Ca²⁺ transient played important roles to determine APDs. This was because [Ca²⁺]_i is an important modulator of cardiac current systems, including I_Ca,L, I_Na-Ca and I_Ks. The "reversed" effects of current injection were even more prominent in our model with a new formulation of I_Ca,L (Ca²⁺-entry dependent inactivation; 2003). Dynamic changes in [Ca²⁺]_i and resultant modulation of current systems play critical roles in cardiac electrophysiology. To progress our understanding on these complex phenomena, further computational as well as experimental investigations are required. [Jpn J Physiol 54 Suppl:S100 (2004)]

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The aim of this study is to elucidate that cardiovascular sympathetic premotor neurons in the medulla oblongata are composed by different kinds of neurons.
We recorded responses of reticulospinal neurons (antidromically activated by electrical stimulation of the dorsolateral funiculus of the spinal cord) and the cutaneous, the cardiac and the renal sympathetic nerves to stimulation of the baroreceptor afferents, warm stimulation of the POAH (49°C), and hypoxia (3%O₂, 97%N₂) in anesthetized and immobilized rabbits.
The activities of non-barosensitive reticulospinal neurons in the rostral ventromedial medulla (Type I) and cutaneous sympathetic nerves were inhibited by warm stimulation of the POAH. However, barosensitive reticulospinal neurons in the rostral ventrolateral medulla (RVLM) and renal sympathetic nerve did not respond to this stimulation. This result suggested that Type I neurons were premotor neurons for cutaneous vasoconstrictors.
Barosensitive reticulospinal neurons in the RVLM were divided into two groups; neurons inhibited by hypoxia (Type II) and ones excited by it (Type III). Because activity of the cardiac sympathetic nerve was inhibited by hypoxia but activity of the renal nerve was excited, it was concluded that neurons of the Type II neurons were premotor neurons for cardiac sympathetic preganglionic fibers. The Type III neurons group might be premotor neurons for vasoconstrictors of visceral organs and/or muscles. [Jpn J Physiol 54 Suppl:S100 (2004)]

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In the heart of adult animals, the hyperpolarization-activated cation channel (HCN4) is exclusively expressed in the pacemaker cells. HCN4 is expressed in the ventricle of fetal heart, and is re-expressed in the ventricle of hypertrophied heart. In order to clarify the transcriptional mechanisms of HCN4 gene, promoter activity was explored using luciferase reporter assay in primary cultured myocytes isolated form embryonic (E12.5) or neonatal (P1-2) rat heart. The hypertrophic stimuli with phenylephrine (50 μM) and endothelin-1 (50 nM) were applied to neonatal myocytes. The promoter deletion analysis showed that proximal –847 bp fragment acted as a minimal promoter both in fetal and neonatal myocytes. The activity of the minimal promoter was significantly higher in embryonic myocyte than in neonatal myocyte, and was insensitive to hypertrophic stimuli. The first intron of hcn4 possesses NRSE, a cis-acting element of neuron-restrictive silencing factor (NRSF). When ~3 kb intron fragment containing NRSE was fused to the minimal promoter of HCN4, promoter activity was highly repressed in neonatal myocytes, whereas the repression was much weaker in fetal myocytes. The hypertrophic stimuli reversed the transcriptional suppression by NRSE-NRSF system. We concluded that multiple cis-acting elements were involved in the transcription of HCN4, and they play different roles in the development and in hypertrophic stimuli. [Jpn J Physiol 54 Suppl:S100 (2004)]

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Abnormalities in cardiac function have been extensively documented in experimental and clinical diabetes. Subcellular remodeling and defects in intracellular Ca²⁺ signaling can contribute to cardiomyopathy resulting in depression of systolic and diastolic function. Moreover diabetes is associated with activation of PKC(Protein Kinase C) signaling pathway account for reduced myocardial gap junctional intercellular communication. Previously we reported that expression of PKCε was enhanced and PKCε-mediated phosphorylation of Cx43(Connexin43) was augmented in association with cell-to-cell decoupling in diabetic heart. It is documented that the diabetic shows hypothyroidism. Thyroid hormones are playing an important role in maintain of cardiac function. Therefor this study was undertaken to examine whether triiodothyronine(T3) treatment in physiological dosage(0.3μg/100g/day) or pharmacological dosage(10μg/100g/day) during chronic stage of diabetes can influence PKC signaling and affect on Cx43 phosphorylation as well as myocardial intercellular electrical coupling. Triiodothyronine showed an ameliorative effect on PKCε-mediated phosphorylation of Cx43 in association with recovery of electrical cell decoupling in the diabetic rat heart. [Jpn J Physiol 54 Suppl:S100 (2004)]

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Nitric oxide (NO) is known to be involved in nicotine-induced increases in cerebral cortical blood flow. To study the exact mechanism(s) of nicotine-induced increases in cerebral blood flow, we measured blood flow in the left hippocampus of the rat using a laser Doppler probe and NO current in the right hippocampus using an NO-selective electrode. Nicotine increased blood flow and decreased NO production in the hippocampus. This indicates that this potentiation is not due to NO-mediated increases in blood flow. Because nicotine has been reported to release adenosine in rat cerebral cortex, we examined the effects of adenosine-related compounds on the responses induced by nicotine in blood flow and NO production. The nonselective adenosine receptor antagonist theophylline attenuated not only the nicotine-induced increases in blood flow but also the nicotine-induced decreases in NO production. The adenosine uptake inhibitor propentofylline, on the other hand, facilitated the nicotine-induced increases in blood flow, but it reduced the nicotine-induced decreases in NO formation. These results suggested that adenosine mediates nicotine-induced increases in hippocampal blood flow. [Jpn J Physiol 54 Suppl:S101 (2004)]

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Background: It has been reported that microbubbles (MB) can be destroyed by ultrasound (US), resulting in a bioeffect, such as angiogenesis. Objective: Our aim is to observe the effect of MB destruction with US on capillary by our intravital CCD videomicroscope. Methods: The superficial capillaries (n=317) of spinotrapezius muscle from 46 WKY rats were exposed to US (2.5 MHz) at mechanical indexes (MI) of 0.1, 1.0, and, 1.9 for 3 min after intraarterial MB injection (Optison). The capillary flow was monitored by the microscope before, during and after US application. Results: We succeeded in capturing real time images of capillary rupture with destruction of MB in 3 capillaries at MI 1.9. The MB destruction with US resulted in flow in which individual RBC halted within capillaries. No-flow after initiation of US irradiation was detected in 6.9%, 21.3%, and 57.6% of all observed capillaries at MI 0.1, 1.0, and 1.9, respectively. In most of capillaries (77.8%) the flow was improved at 15min, while in other fraction of capillaries (22.2%) flow disturbance remained at that time, followed by slow flow recovery. Exposure to US alone did not induce no-flow. Conclusions: US exposure to MB in capillaries transiently resulted in MI-dependent microvascular damage including land-mine like capillary rupture. [Jpn J Physiol 54 Suppl:S101 (2004)]

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A Rho-kinase (ROK)-mediated Ca²⁺-sensitization plays an important role in the pathogenesis of abnormal vascular smooth muscle (VSM) contraction such as vasospasm. Previously we reported that sphingosylphosphorylcholine (SPC) induced ROK-mediated Ca²⁺-sensitization in VSM contraction via the activation of Src family tyrosine kinase (Src-TKs) and that SPC levels were elevated in vasospasm patients. However, the signal transduction mechanism by which Src-TKs can activate ROK is unknown. To identify the downstream signaling molecules of Src-TKs, we screened the molecules in which SPC and Src-TKs inhibitors can modulate the tyrosine phosphorylation in porcine coronary and rabbit mesenteric arteries, using anti-phosphotyrosine antibody (RC20). Arterial strips were stimulated with SPC to induce contraction and rapidly frozen-fractured at various time points and subjected to the immunoblot analysis against RC20. SPC modulated tyrosine phosphorylation in several molecules including p60, and PP2 and eicosapentaenoic acid (EPA), Src-TKs inhibitors, diminished the effect of SPC on tyrosine phosphorylation and the accompanying contraction. To identify those molecules, RC20-immunoprecipitated samples were separated by SDS-PAGE, transferred to PVDF membrane. The protein band correspond to p60 was excised, digested by Achromobacter Protease I and subjected to MALDI TOF-MS analysis for peptide mass fingerprinting. The result showed two tyrosine-phosphorylated proteins as the candidates for p60. [Jpn J Physiol 54 Suppl:S101 (2004)]

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To study the development of the aortic baroreceptor reflex(ABR)under conditions of microgravity, we examined the fiber composition of the left aortic nerve(LAN) from rats aged 25 days raised in microgravity on the Space Shuttle Columbia(FLT group) for 16 days. Two ground control groups (same cage as that for FLT and commercial cage) were compared with FLT. We have confirmed the alteration of the LAN under microgravity, to study their relationship with the functional characteristics of the ABR in anesthetized rats(urethane, 1.2-1.8 g/kg). It was difficult to obtain both histologic and functional data from the individual animals because of the some limitations in the space experiment. Thus, we had original idea to investigate the relationship between percentile proportion of unmyelinated fibers(UMF) and indexes of the ABR at the peak increase in arterial pressure; we calculated averages of factors obtained by each experiment to normalize the characteristics of the three groups. These results showed that the FLT group indicated the lower peak increase in mean arterial pressure for a given dose of phenylephrine and the fewer aortic UMF, the fewer UMF leaded the lower aortic afferent sensitivity, and the fewer UMF related with the lower index of baroreflex sensitivity. [Jpn J Physiol 54 Suppl:S101 (2004)]

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We have recently developed a method for determination of the time courses of the junctional sarcoplasmic reticulum(JSR) Ca²⁺replenishment and release in heart muscle by examining the time courses of the mechanical restitution after varying magnitudes of twitch contractions . To study Ca²⁺ handling by the JSR in the heart muscle of the hypertensive rat(SHR), we applied this method to the heart muscle of the SHR and the control(WKY).The mechanical restitution consisted of a pre-twitch latency period followed by a rapid and a subsequent much slower restitution of twitch force in both the SHR and the WKY. The rate of rapid restitution was independent of the magnitude of the preceding twitch in the WKY and the SHR, besides that it was smaller after the rested state twitch than the smaller twitch in the SHR. The rate of rapid restitution was similar in the WKY and the SHR unless the magnitude of the preceding twitch in the latter was smaller than about 1/2 of the rested state twitch. Based on this finding, the functions G(t) and H(t) , representing the time courses of the JSR Ca²⁺ replenishment and release, respectively, were derived graphically from a family of the mechanical restitution curves for the WKY and the SHR. The result suggests that the G(t) is a monotonically increasing function with time,while the H(t) a sigmoid function, and that the JSR Ca²⁺ content immediately after the rested state twitch is much smaller in the SHR than the WKY. [Jpn J Physiol 54 Suppl:S102 (2004)]

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Introduction: Catechol-O-methyltransferase (COMT) plays an important role for clearance of elevated catecholamine concentrations. Although experimental ischemic models clearly demonstrate marked accumulation of catecholamine in the heart, there are few reports on the physiological role of COMT activity for clearance of norepinephrine (NE) accumulation evoked by ischemia. We examined contribution of COMT to change in myocardial NE levels during myocardial ischemia and reperfusion. Methods: Acute myocardial ischemia was induced in anesthetized rabbits by a 60 min occlusion of the main coronary artery. We implanted a microdialysis probe into the left ventricular and measured myocardial interstitial NE and its metabolites levels in anesthetized rabbits. As an index of COMT activity, dialysate normetanephrine (NMN) levels were measured. We introduced myocardial ischemia by 60-min occlusion of the main coronary artery. The effects of entacapone (COMT inhibitor 10mg/kg, i.p.) on the ischemia-induced dialysate NE and NMN levels were examined. Results: Acute myocardial ischemia increased dialysate NE and NMN levels. Inhibition of COMT activity by entacapone augmented the ischemia-induced NE accumulation at 30-45 min of the coronary occlusion (vehicle: 27288 ± 7231, entacapone: 46146 ± 5979 pg/ml). Ischemia-induced NMN production was suppressed by entacapone (vehicle: 2023 ± 339, entacapone: 557 ± 136 pg/ml). Conclusion: In the ischemic heart, COMT activity contributes to removal of accumulated NE. [Jpn J Physiol 54 Suppl:S102 (2004)]

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The outward current of I_K1 shows significant amplitude in the voltage range near the reversal potential and thereby causes rapid repolarization at the final phase of action potentials. However, the mechanism that produces the outward I_K1 is not understood. The currents generated by expressing the strong inward rectifier K⁺ channel gene Kir2.1 resemble I_K1 under the whole-cell recordings. In this study, we recorded currents from inside-out patches of 293T cells that express Kir2.1 and studied the blockage of the currents by cytoplasmic polyamines. The outward current-voltage relationships, obtained with 5-10 μM spermine (spm) or 10-100 μM spermidine (spd), were similar to that of I_K1, while they exhibited a plateau or a double-hump shape at lower spm/spd concentrations. There were extra conductances in the chord conductance-voltage relationships, which could not be described by the Boltzmann relations fitting the major part of the relationships. The extra conductances were quantitatively explained by a model that considered two populations of channels, which were blocked by polyamines in either a high-affinity mode (Mode 1 channel) or a low-affinity mode (Mode 2 channel). The estimated proportion of Mode 1 to total channels was 0.9 with 0.1-10 μM spm and between 0.75 and 0.9 when spm and spd coexisted. Our results suggest that the outward current of I_K1 is primarily generated by the Mode 2 channels. Polyamines may regulate I_K1, not only by blocking the channels but also by modifying the proportion of channels that show different sensitivities to the polyamine block. [Jpn J Physiol 54 Suppl:S102 (2004)]

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We have previously reported that high Ca²⁺ infusion produces a Ca²⁺-overloaded contractile failure in rat hearts associated with a parallel downward shift of the linear relation between left ventricular (LV) myocardial oxygen consumption per beat (VO₂) and systolic pressure-volume area (PVA) (VO₂-PVA relation), linking to proteolysis of a cytoskeletal protein, α-fodrin. We hypothesized that intracoronary infusion with a β-adrenergic receptor agonist, dobutamine causes a similar contractile failure after its clearance. To test this hypothesis, we investigated LV mechanical work and energetics in cross-circulated excised rat hearts. After control volume loading run (control vol-run), two different runs during dobutamine infusion were performed. After stopping dobutamine infusion, back control vol-run was performed. The curved LV end-systolic pressure-volume relation obtained in back control vol-run shifted downward from that in control vol-run. The VO₂-PVA relation also shifted downward; the VO₂ intercept significantly decreased (n=7). In these hearts, we found a proteolysis of α-fodrin in the hearts underwent intracoronary infusion with dobutamine. The result indicated that LV mechanical work and energetics were suppressed after clearance of dobutamine similar to high Ca²⁺, though they were enhanced during dobutamine infusion. The suppression on mechanoenergetics in these stunned hearts is linked to a proteolysis of α-fodrin as in Ca²⁺-overloaded contractile failing hearts. [Jpn J Physiol 54 Suppl:S102 (2004)]

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To examine whether a single cardiomyocyte preparation retains the mechanical characteristics of ventricle, we measured the contractile function of a single rat ventricular myocyte under a wide range of loading conditions using a force-length measurement system implemented with adaptive control methods. A pair of carbon fiber was used to clamp the myocyte. One fiber was stiff, serving as a mechanical ground, while the bending motion of the other compliant fiber was monitored for force-length measurement. Furthermore, by controlling the position of the compliant fiber using a piezo-electric translator based on adaptive control strategy, we could change both preload and afterload dynamically during contractions. Under isometric condition, peak developed force reached 41.6±2.7 mN/mm² (mean±S.E.M., n=17), and under unloaded condition, maximal shortening velocity was 106±5.3 μm/s (n=13). When we simulated physiological loading conditions consisting of an isometric contraction followed by shortening and relaxation, the average work-output was quite close to that expected from the total external work of the left ventricle. The upper left corners of force-length loops obtained under these conditions distributed along a straight line, analogous to the end-systolic pressure-volume relation of the ventricle. [Jpn J Physiol 54 Suppl:S103 (2004)]

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The response of mitochondrial NADHm to the increase in workload is still controversial. We measured NADHm fluorescence in isolated guinea pig ventricular myocytes stimulated at various stimulus frequency at 37°C. The NADHm fluorescence was excited at 340/20 nm, and the emission was measured at 460/50 nm. Simultaneously, the longer axis of the cell was measured by a liner image sensor with light at 540/30 nm. The membrane potential was recorded using amphotericinB perforated patch. When the positive staircase was obvious on changing the stimulus frequency from 0.1 Hz to 3.3 Hz, NADHm initially decreased by about 15.6% of control and subsequently increased to maximum levels (158% of control). When the stimulus frequency was returned to 0.1 Hz, a transient increase in NADHm was observed before returning to the control level. After treatment with ruthenium red, an inhibiter of mitochondrial calcium uniporter, this increase of NADHm during stimulating at 3.3 Hz was attenuated. We measured mitochondrial calcium (Ca²⁺m) using Rhod-2AM (excitation: 540/25 nm, emission: 590/55 nm) and the cell length at 710/75 nm simultaneously with the same stimulating protocol. Beat to beat Ca²⁺m change was recognized, and Ca²⁺m increased during stimulating at 3.3 Hz and returned to the control level at 0.1 Hz. The initial decrease of NADHm might be attributed to the increase of ADP ('respiratory control'). The delayed increase of NADHm suggests that Ca²⁺m should stimulate NADH production in response to the increased workload ('parallel activation'). [Jpn J Physiol 54 Suppl:S103 (2004)]

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Mouse embryonic stem (mES) cells have abilities to differentiate into various types of the cells, including cardiomyocytes. In this study we examined Ca²⁺ signaling pathways in mES cells for excitation-contraction (E-C) coupling during the differentiation into cardiomyocytes. To identify the cardiac precursor cells, we used the mES cells transfected with the vector containing GFP driven by mouse cardiac α-actin promoter. Caffeine effects on [Ca²⁺]_i were observed in GFP-positive precursor cells cultured for longer than 5 days without leukemia inhibitory factor and these effects increased during the differentiation. A depolarization induced elevation of [Ca²⁺]_i was observed in 10%, 13%, 25% and 46% of 4-day, 5-7 day, 12-14 day and 24-27 day differentiated cells, respectively. In whole cell patch clamp experiments, T-type but not L-type Ca²⁺ currents could be recorded in the precursor cells at 12-day stages. In 19-day or beating cells, L-type Ca²⁺ currents were observed. The expressions of mRNAs for RyR2 and T-type Ca²⁺ channel were detected from 4-day differentiated cells, but mRNAs for L-type Ca²⁺ channel could be detected from 7-day differentiated cells. The expression levels of these genes increased during the differentiation. From these results we conclude that main Ca²⁺ entry pathways switch from T-type Ca²⁺ channel to L-type one during the differentiation, which might contribute to E-C coupling. [Jpn J Physiol 54 Suppl:S103 (2004)]

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Background: Because of the thickness and the three-dimensionally complex structures of the left ventricle (LV), it has been difficult to observe how crossbridge dynamics correlate with LV performance in a beating heart preparation using a second-generation synchrotron radiation. Recently we confirmed the possibility to observe the transmural crossbridge (CB) dynamics (CBD) by analyzing the reflection of the beating LV free wall associated with transmural variations of regional myofilament orientations using a third generation synchrotron facility. Purpose: To reveal transmural difference of crossbridge dynamics in the beating LV. Methods: We paced isolated crystalloid perfused isovolumically beating rat hearts (n=5) at 2Hz. We recorded x-ray diffraction images of LV epimyocardial and midmyocardial regions at SPring-8 with LV pressure (LVP) and analyzed transmural CBD utilizing transmural variations of regional myofilament orientations. Results: During contraction, normalized LVP and transmural CBDs temporally harmonized well each other. However during relaxation, the CB decay of midmyocardium was faster, but that of epimyocardium later than LVP. Conclusion: The transmural CBD and LVP development well harmonized temporally during systole while their decay during diastole disaccord with each other. [Jpn J Physiol 54 Suppl:S103 (2004)]

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The effects of extracellular ATP on β-adrenergic activation of CFTR Cl current (I_Cl,PKA) were studied in guinea-pig ventricular cells. The cells were initially exposed to 0.02-1 μM isoproterenol (ISO) for ~3 min to activate I_Cl,PKA, and then to 1-100 μM ATP in the presence of ISO. ATP was found to potentiate I_Cl,PKA, in most cells examined. In about 2/3 of them, however, the potentiation was preceded by an inhibition, I_Cl,PKA changing in a biphasic manner. The initial inhibition was due to stimulation of P1-purinoceptor by ATP, since the inhibition was attenuated by the blockers of this receptor type. With 50 μM ATP, the potentiation, on average, resulted in a 1.3 fold increase of the Cl conductance activated by ISO alone (0.02-1 μM). The effects of ADP and ATPγS on I_Cl,PKA were similar to those of ATP, while AMP and adenosine never potentiated I_Cl,PKA. Thus the potentiation was attributed to a stimulation of P2-purinoceptors. PDBu (0.5 μM), an activator of PKC, facilitated I_Cl,PKA, and in the presence of PDBu, ATP did not further potentiate I_Cl,PKA. When BIM (0.2 μM), an inhibitor of PKC, was present, ATP did not facilitate I_Cl,PKA. These findings suggested involvement of PKC in the observed ATP action. When ATP was removed in the presence of ISO, the potentiated I_Cl,PKA decreased (recovered) only slowly, and, if ATP was reapplied during this slowly recovering phase, the subsequent current potentiation was weak. Thus the stimulation of P2-purinoceptors by ATP facilitates the β-adrenergic activation of I_Cl,PKA through PKC activation, and this potential appears to persist for several min after removal of ATP. [Jpn J Physiol 54 Suppl:S104 (2004)]

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The dynamical mechanisms of early afterdepolarizations (EADs) were investigated by bifurcation analyses, as well as numerical simulations, using a mathematical model of human ventricular myocytes. On the basis of experimental data, we have developed a human ventricular model as a modified version of previous models. The stability of equilibrium points (EPs), bifurcation structures, and oscillation dynamics of the model system was determined as functions of the L-type Ca²⁺ channel conductance (gCa,L), delayed-rectifier K⁺ channel conductance (gKr, gKs), or noninactivating sodium channel conductance (gNass). We first examined how EADs appear in the normal system and in long QT syndromes, which were simulated by decreasing gKs (LQT1), decreasing gKr (LQT2), or increasing gNass (LQT3). EADs appeared in the vicinity of Hopf bifurcation (HB) points, when gCa,L was increased, or when gKs and/or gKr was decreased. The threshold (critical gCa,L value) for EADs to appear during gCa,L increases was much lower in the LQTs than in the normal system; the critical gK was much higher in the LQTs. We further identified the gating variable of the slowly-activating delayed-rectifier K⁺ current (n) as a slow subsystem, exploring the effects of a parameter n (gKs) on the bifurcation structures of the fast subsystem (slow-fast decomposition analysis). Facilitation of the EAD generation in the LQTs appeared to be due to the shift in HB values toward higher n (gKs) values. [Jpn J Physiol 54 Suppl:S104 (2004)]

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We have shown that the dynamics of crossbridge cycling can now be determined in beating hearts in situ and in real time using high flux synchrotron radiation at SPring-8 and x-ray diffraction techniques. Mass transfer of myosin to actin during contraction was inferred from the change in diffraction intensity ratio (intensity of 1,0 reflection / intensity of 1,1 reflection) obtained from the lattice arrangements of myosin (thick) and actin (thin) filaments in myofibers between diastole and systole. Others have suggested that myofilament spacing might influence the probability of crossbridge formation. In this study we examined for the first time, if myosin lattice spacing (d_1,0 spacing between myosin planes) is regulated to maintain constant lattice volume and how mass transfer is related to d_1,0 spacing during the cardiac cycle in different regions of the left ventricle. Our results show that crossbridge formation was essentially complete within the isovolumic contraction phase, and that while mass transfer was proportional to left ventricle pressure in non-ischemic hearts, this relation deteriorated in ischemic regions of the heart after coronary occlusion. This suggests that diffraction intensity ratio change is more sensitive to regional contractility than global contractility indices. As lattice spacing was significantly larger during contraction than ventricular filling (at the same volume) we suggest that constant lattice volume is not maintained during dynamic contractions. [Jpn J Physiol 54 Suppl:S104 (2004)]

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To investigate whether thyroid hormone modulates electrical properties of atrial myocytes, electrocardiogram (ECG), action potentials (APs), and ionic currents were measured. Male Sprague-Dawley rats were randomly divided into control and T₄-treated groups at 6 weeks of age. L-thyroxine (500 μg/kg body weight) was daily injected into peritoneal cavity for 14 days (T₄-treated rats) and the same volume of saline was daily injected in control rats. ECG signals were recorded using apex-base lead. APs, voltage-dependent Na⁺ and L-type Ca²⁺ channel current (I_Na and I_Ca(L)), inwardly rectifying K⁺ channel current (I_K1), transient outward K⁺ channel current (I_to), and delayed rectifier K⁺ channel current (I_K(delay)) were measured using patch-clamp techniques. T₄-treatment significantly changed electrical properties in rat atrial myocytes including the increase in cell membrane capacitance, the decrease in input resistance, and the shortening of AP duration. ECG revealed sinus tachycardia in T₄-treated rats. Voltage-clamp recording revealed the current densities of I_Na and I_K1 in T₄-treated atrial myocytes did not differ from those in control cells. However, the current density of I_Ca(L) was significantly decreased and the current density I_K(delay) was significantly increased in T₄-treated rats. Thus, thyrotoxicosis could induce the shortening of AP duration by alterations in the current densities of both I_Ca(L) and I_K(delay) in rat atrial myocytes. [Jpn J Physiol 54 Suppl:S104 (2004)]

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The dynamic computer simulation is essential for quantitative understanding of integrated mechanisms underlying various cell functions. The two-dimensional models of action potential propagation successfully demonstrated the basic mechanisms of re-entry. However, the gap junction conductance is regulated by, for example, the intracellular Ca²⁺, pH, and protein kinase. Thus, to examine the physiological and pathophysiological conditions, we need to consider many aspects such as intracellular Ca²⁺ homeostasis, energy metabolism, and so on. To evaluate the effect of varying electrical coupling on the electro-mechanical properties of myocytes, we coupled individual ventricular cell model. When the two ventricular cell models were connected with the conductance over 7 nS, the action potential of the 1st cell propagated to the 2nd cell with a clear delay, though the repolarizing phase was almost synchronized. Under this condition, the action potential of the 1st cell showed a marked downward notch after the peak. The amplitude of Ca²⁺ current in the 1st cell was increased during the notch because of larger driving force, so that the Ca²⁺ transient and contraction were increased. Thus, the 1st cell produced a larger contraction than the 2nd one. Increasing the number of coupled cells from 2 to 5, a larger gap junction conductance was needed to propagate the action potential. The dynamic regulation of conductance described in the literature should be applied to the gap junction model. [Jpn J Physiol 54 Suppl:S105 (2004)]

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Leukocyte adhesion and rolling on endotherial surface of venules were frequently obseved under an intravital microscope. Adhesive molecules of endothelium, platelet and leukocyte relate to these behavior. However hemodynamical facter as shear stress to leukocyte on endtherium also relate to these behavior. Because there was a little imformation of hemodynamical facters of leukocyte behavior, we studied relationship between these behavior and blood flow velocity in venules during acute hemodynamical changes in the microcirculation. Anethetized Wistar rats were used under spontaneous respilation. The mesentery was draped on a observastion window on the stage of a inverted microscope equiped with a video camera and an optic fiber system for measuring blood flow velosity in a microvessel. The obtained image were recorded by a video-cassette recorder and analyzed by using a computer-assisted image analysis system. The inner diameter and leukocyte number were measured on the digitalized video images using the public domain NIH image program. Blood flow velocity was measeurd using a dual-slit photomeric method with cross-correlatin function by a computer-assist. When blood flow velocity in venules decreased from physiologica condition, leukocyte number incresead, but rolling velocity of luekocye did not changed. When blood flow velocity in venules increased, leukocyte number decreased and rolling velocity of leukocyte incresead. This increasing of rolling velocity was steplike form at high shear rate condition in venule. [Jpn J Physiol 54 Suppl:S105 (2004)]

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Interstitially administered colloidal particles are known to accumulate in the regional lymph nodes. Recently, radiolabeled colloids, such as ^99mTc-Sulfer colloid and ^99mTc-Rhenium colloid, are used for lymphoscintigraphy and sentinel node navigation surgery. Accumulation of interstitially injected colloidal particles in the lymph nodes involves the following three steps; uptake of the colloids at the initial lymphatics, transport of the colloids through the collecting lymphatics, and retention of the colloids within the lymph nodes. In this study, we focused on barrier function of the regional lymph nodes in order to investigate characteristics of colloidal retention within the lymph nodes. Centripetal and retrograde cannulations were performed into the popliteal afferent and efferent lymph vessels in Japan white rabbits, respectively. The lymph nodes were selectively perfused (injection rate: 0, 0.74, 1.47, 3.68, 7.35 and 14.7 ml/h) with artificial lymph fluid (HBSS with 2.6% BSA) in the absence or presence of fluorescently labeled microsphere. More than 80% of the volume of the fluid injected into the afferent lymphatic was collected from the efferent one. No microsphere of 2.0 μm in diameter injected into the afferent lymphatic was appeared in the efferent one. On the other hand, microspheres of 0.2 μm in diameter injected could be observed in the efferent lymph vessel. These results suggest that this preparation is useful to evaluate physiological and pathophysiological function of the lymph nodes in-vivo. [Jpn J Physiol 54 Suppl:S105 (2004)]

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